703360

antibody from Invitrogen Antibodies

Targeting: H3-3B H3.3B, H3F3B

Western blot (Supportive data in Antibodypedia)

Chromatin Immunoprecipitation (Supportive data in Antibodypedia)

Antibody Data

Product number

703360

Provider

Invitrogen Antibodies

Product name

H3.3 G34R oncohistone mutant Recombinant Rabbit Monoclonal Antibody (4H1L8)

Antibody type

Monoclonal

Antigen

Synthetic peptide

Reactivity

Human

Host

Rabbit

Isotype

IgG

Antibody clone number

4H1L8

Vial size

100 µg

Concentration

0.5 mg/mL

Storage

Store at 4°C short term. For long term storage, store at -20°C, avoiding freeze/thaw cycles.

Western blot

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot analysis was performed on different concentrations of H3.3 recombinant protein (wild-type) and H3.3 G34R recombinant protein (mutant). The blot was probed with Anti-H3.3 G34R Recombinant Rabbit Monoclonal Antibody (Product # 703360, 1:500 dilution) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.25 µg/mL, 1:4000 dilution). A 17 kDa band corresponding to H3.3 G34R was observed only in the mutant recombinant protein.

Chromatin Immunoprecipitation

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Enrichment of overexpressed histone H3.3 G34R protein at specific gene loci using Anti-H3.3 G34R Antibody: Chromatin Immunoprecipitation (ChIP) was performed using Anti-H3.3 G34R Recombinant Rabbit Monoclonal Antibody (Product # 703360, 4 µg) on sheared chromatin from 2 million control (a; vector only transfected) or histone H3.3 G34R overexpressing (b) HEK cells using the MAGnify ChIP System kit (Product # 49-2024). Normal Rabbit IgG was used as a negative IP control. The purified DNA was analyzed by qPCR with PCR primer pairs over the GAPDH gene (Active) and SAT2 satellite repeats (Inactive). A schematic diagram of the GAPDH gene is shown on top of the figure. Data is presented as fold enrichment of the antibody signal versus the negative control IgG using the comparative CT method.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Recombinant Monoclonal H3.3 G34R antibody (Product # 703360) was tested in a native ChIP experiment with ~1x10^6 HEK-293 cells containing a spike-in of the SNAP-ChIP OncoStat panel (EpiCypher®, 19-2001) of DNA-barcoded nucleosomes with either wild-type H3.3 or one of the seven distinct H3.3 oncogenic mutations (X-axis). Specificity (left Y-axis) was determined by quantitative real-time PCR (qPCR) for the barcodes corresponding to each nucleosome in the panel. Black bar represents antibody efficiency (right Y-axis; log scale) and indicates the percentage of the SNAP-ChIP H3.3 G34R nucleosome target immunoprecipitated relative to its Input. H3.3 G34R antibody exhibited <5% cross-reactivity outside H3.3 G34R and immunoprecipitated the target with ~2% efficiency. Error bars represents mean 177 SEM from N=3 independent ChIP experiments.